Single molecule force spectroscopy of single stranded DNA binding protein and Rep helicase

Single-stranded DNA binding (SSB) proteins are essential accessory proteins that protect single-stranded DNA (ssDNA) during genome maintenance. Escherichia coli SSB is a prototypical homo-tetrameric SSB protein that can wrap up to 65 nucleotides of ssDNA in one of its binding modes. Here we present mechanical studies of E. coli SSB bound to ssDNA using high-resolution optical tweezers. This method allows us to probe the interaction of individual SSBs to ssDNA in real time, with nanometer resolution. By detecting directly the wrapping of ssDNA by a single protein, we are able to characterize the thermodynamics and kinetics of nucleoprotein complex formation. Mechanical pulling of ssDNA in the presence of SSB reveals that the protein condenses ssDNA in the force range 0-10pN and that tension can be used to modulate the ssDNA wrapping state of SSB. Measurements of SSB kinetics indicate that SSB-ssDNA complex formation occurs in a two-step process consisting of a diffusion-limited binding step in which the protein associates weakly with its substrate, followed by a fast wrapping step in which ssDNA is condensed. We also quantify how tension modulates the ssDNA wrapping state of SSB, revealing features of the energy landscape for SSB-ssDNA interactions. Lastly, we carried out measurements of SSB interaction with long ssDNA binding substrate as a function of mechanical force. The data indicate that SSBs bound to longer stretches of ssDNA bound much tighter, probably due to nucleoprotein filament formation. And we have evidence that SSB can bind to ssDNA in intermediate wrapping states and are transiently wrapping and unwrapping from their substrates. In addition we present for the first time study on the conformational control of Rep helicase using an optical trap. We found that crosslinking-mediated conformational arrest of a dynamic subdomain in so-called “closed” orientation converted the Superfamily I (SFI) helicase, Rep, from a very poor DNA helicase into a powerful motor protein with a highly processive DNA unwinding activity. In contrast, the wild type Rep helicase cannot efficiently unwind DNA over 18 bp in vitro. A single Rep-X (cross-linked Rep) molecule can processively unwind DNA up to 4 kbp and generate forces in excess of 40 pN, making it the most powerful helicase known. The same modification on the related PcrA helicase produced the same activity increase characteristics strengthening the possibility of widespread application of this conformational control technique. Thus our results directly answer the question of the role of different conformations observed in SF-I helicase crystal structures and offer a mechanism for how partner proteins in vivo may regulate helicase function via conformational control.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Physics

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2012

Author dc:creator

• Khafizov, Rustem

Contributors dc:contributor

• Chemla, Yann R.
• Ha, Taekjip
• Stack, John D.
• Oono, Yoshitsugu

• Optical Trap
• Single-stranded DNA binding protein
• Single-stranded DNA binding (SSB)
• Rep helicase
• single molecule

Statement dc:rights

• Copyright 2012 Rustem Khafizov

Language dc:language

en

Handle dc:identifier

http://hdl.handle.net/2142/34441

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/34441